Rapid high throughput assay for fluorimetric detection of doxorubicin--application of nucleic acid-dye bioprobe

A double stranded DNA based fluorescence bioprobe for anticancer agent (doxorubicin) detection is described. This method provides a new way for sensitive DNA/drug interaction study by a homogeneous assay. The probe employs the long-wavelength intercalating fluorophore TOTO-3^® (TT3). The anticancer agent, doxorubicin, which interacts with the DNA-TT3 complex, was indirectly measured by the decrease in the fluorescence intensity. Various oligonucleotides with different sequences were examined. Doxorubicin has preference for the oligonucleotide 5′AGCACG3′. Enhanced fluorescence observed for the TT3 intercalation with this oligonucleotide makes the DNA-dye complex a suitable bioprobe for doxorubicin detection by competitive assay. A home-built CCD camera setup was applied along with 384 well plate assay format for high throughput fluorescence imaging. The detection limit can be as low as 25 ng mL⁻¹ with an upper limit of 100 μg mL⁻¹. The recovery test with spiked serum sample shows that this method can be a potential routine method for therapeutic drug monitoring (TDM).     

Rapid,single-step nucleic acid detection

A rapid detection method for nucleic acid based on bioluminescence resonance energy transfer (BRET) from the luminescence donor Renilla luciferase to an acceptor quantum dot upon oligonucleotide probe hybridization has been developed. Utilizing a competitive assay, we detected the target nucleic acid by correlating the BRET signal with the amount of target present in the sample. This method allows for the detection of as little as 4 pmol (20 nM) of nucleic acid in a single-step, homogeneous format both in vitro in a buffer matrix as well as in a cellular matrix. Using this method, one may perform nucleic acid detection in as little as 30 min, showing much improvement over time-consuming blotting methods and solid-phase methods which require multiple wash steps to remove unbound probe. This is the first report on the use of quantum dots as a BRET acceptor in the development of a nucleic acid hybridization assay. An erratum to this article can be found at     

Planar waveguides for ultra-high sensitivity of the analysis of nucleic acids

In the first part of this paper, the need for analytical techniques capable of highly parallel and sensitive nucleic acid analysis, with the capability of achieving very low limits of detection (LODs) and of resolving small differences in concentration, is described. Whereas the requirement for performing simultaneously multi-analyte detection is solved by the approach of nucleic acid microarrays, requirements on sensitivity can often not be satisfied by classical detection technologies. Inherent limitations of conventional fluorescence excitation and detection schemes are identified, and the implementation of planar waveguides as analytical platforms for nucleic acid microarrays, with fluorescence excitation in the evanescent field associated with the guided excitation light, is proposed. The relevant parameters for an optimization of sensitivity are discussed.In the second part of this paper, the specific formats of our planar waveguide platforms, which are compatible with established industrial standard formats allowing for integration into industrial high throughput environments, are presented, as well as the dedicated optical system for fluorescence excitation and detection that we developed. In a direct comparison with a state-of-the-art scanner, it is demonstrated that the implementation of genomic microarrays on planar waveguide platforms allows for unprecedented, direct detection of low-abundant genes in limited amounts of sample. Otherwise, when using conventional fluorescence excitation and detection configurations, the detection of such low amounts of nucleic acids requires massive sample preparation and signal or target amplification steps.     

Fluorimetric determination of nucleic acid using the enhancement of terbium-gadolinium-nucleic acid-cetylpyridine bromide system

It is found that the fluorescence intensity of Tb-cetylpyridine bromide (CPB)-nucleic acid system can be enhanced by La³⁺, Gd³⁺, Lu³⁺, Sc³⁺ and Y³⁺, of which Gd³⁺ has the greatest enhancement. The experiments indicate that under the optimum condition, the fluorescence intensity of the system is in proportion to the concentration of nucleic acids in the range from 9×10⁻⁸ to 1×10⁻⁵ g ml⁻¹ for yeast RNA, from 1×10⁻⁷ to 1×10⁻⁵ g ml⁻¹ for fish sperm DNA. The detection limits are 3.2 and 4.1 ng ml⁻¹, respectively. This method has been used satisfactorily for the determination of both synthetic and actual samples. In comparison with most fluorescence method for the determination of nucleic acids, this method has higher sensitivity and stability.     

Capillary waveguide nucleic acid based biosensor

Micro-capillaries are finding increasing utility in the development of portable analytical sensors. We present design guidelines for optimizing the collection of free propagating fluorescence for capillary waveguide sensors used in the detection of nucleic acids. A dual function integrated opto/fluid connector is also described. Evanescent wave excitation of the coating layer containing a DNA probe is achieved by using a fiber optic ring arrangement for coupling light directly into the capillary wall. The central part of the connector is used for injecting a DNA or RNA target into the capillary channel. In situ hybridization has been used to detect target molecules at a concentration of 30 pg ml⁻¹. The sensor can be regenerated for repeated detection of DNA or RNA targets.     

On-chip multiplexed solid-phase nucleic acid hybridization assay using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer

A microfluidic based solid-phase assay for the multiplexed detection of nucleic acid hybridization using quantum dot (QD) mediated fluorescence resonance energy transfer (FRET) is described herein. The glass surface of hybrid glass-polydimethylsiloxane (PDMS) microfluidic channels was chemically modified to assemble the biorecognition interface. Multiplexing was demonstrated using a detection system that was comprised of two colors of immobilized semi-conductor QDs and two different oligonucleotide probe sequences. Green-emitting and red-emitting QDs were paired with Cy3 and Alexa Fluor 647 (A647) labeled oligonucleotides, respectively. The QDs served as energy donors for the transduction of dye labeled oligonucleotide targets. The in-channel assembly of the biorecognition interface and the subsequent introduction of oligonucleotide targets was accomplished within minutes using a combination of electroosmotic flow and electrophoretic force. The concurrent quantification of femtomole quantities of two target sequences was possible by measuring the spatial coverage of FRET sensitized emission along the length of the channel. In previous reports, multiplexed QD-FRET hybridization assays that employed a ratiometric method for quantification had challenges associated with lower analytical sensitivity arising from both donor and acceptor dilution that resulted in reduced energy transfer pathways as compared to single-color hybridization assays. Herein, a spatial method for quantification that is based on in-channel QD-FRET profiles provided higher analytical sensitivity in the multiplexed assay format as compared to single-color hybridization assays. The selectivity of the multiplexed hybridization assays was demonstrated by discrimination between a fully-complementary sequence and a 3 base pair sequence at a contrast ratio of 8 to 1.     

石墨烯材料在蛋白质和核酸分离/检测中的应用进展

石墨烯是碳材料家族新兴成员之一,因其具有化学性质稳定、良好生物兼容性、催化性、零带隙、特殊的电子能带等特点而受到科学家们广泛关注。 石墨烯特殊的六元环基底平面对很多分子、离子具有亲和性,可通过非共价键或共价键将目标物质富集于表面,大多数情况下被吸附的物质在特定条件下易解吸且不会引起其结构和性质的改变。 石墨烯因而在生物成像、生物医学、光催化、生物传感、药物载体和释放、 环境等方面应用前景广阔。 本文主要论述石墨烯和它的衍生物在生命大分子( 蛋白质和核酸) 分析化学方面的应用,并展望了其发展趋势。     

Sequence-specific detection of nucleic acids utilizing isothermal enrichment of G-quadruplex DNAzymes

G-quadruplex DNAzymes are peroxidase-like complexes formed by nucleic acid G-quadruplexes and hemin. Various chemical sensors and biosensors have been developed, based on such DNAzymes. Here we report a novel, specific nucleic acid detection method utilizing the isothermal amplification strategy of G-quadruplex DNAzymes. In this method, an unlabeled oligonucleotide probe was used. The probing sequence of the oligonucleotide was in the form of a stem-loop structure. A G-rich sequence, containing three GGG repeats, was linked to the 5′-end of the stem-loop structure. In the presence of target, the probing sequence hybridized to the target, and a G_n (n ≥ 2) repeat was extended from its 3′-end. This G_n repeat, together with the three GGG repeats at the 5′-end, folded into a G-quadruplex, and displayed enhanced peroxidase acitivity upon hemin binding. Utilizing the dynamic binding interaction between the probe and its target, the enrichment of G-quadruplex DNAzymes was achieved. Using this method, simple, rapid and cost-effective nucleic acid detection could be achieved. This method displayed high target-length tolerance and good detection specificity; one-base mismatch could be judged easily, even by visual inspection. This method may be used as an auxiliary tool for amplified detection of specific DNA targets in some situations, in which isothermal detection is desirable.     

Fungal pathogenic nucleic acid detection achieved with a microfluidic microarray device

Detection of polymerase chain reaction (PCR) products obtained from cultured greenhouse fungal pathogens, Botrytis cinerea and Didymella bryoniae has been achieved using a previously developed microfluidic microarray assembly (MMA) device. The flexible probe construction and rapid DNA detection resulted from the use of centrifugal pumping in the steps of probe introduction and sample delivery, respectively. The line arrays of the oligonucleotide probes were “printed” on a CD-like glass chip using a polydimethylsiloxane (PDMS) polymer plate with radial microfluidic channels, and the sample hybridizations were conducted within the spiral channels on the second plate. The experimental conditions of probe immobilization and sample hybridization were optimized, and both complementary oligonucleotides and PCR products were tested. We were able to achieve adequate fluorescent signals with a sample load as small as 0.5 nM (1 μL) for oligonucleotide samples; for PCR products, we achieved detection at the level of 3 ng.     

四磺化铁酞菁-阿尔新蓝8GX-对-羟基苯乙酸-H2O2催化荧光反应体系在核酸定量中的应用

阳离子荧光染料阿尔新蓝8GX与阴离子荧光染料四磺化铁酞菁发生缔合作用，使四磺化铁酞菁的荧光猝灭，当核酸存在时，削弱了离子缔合作用使四磺化铁酞菁的荧光恢复。荧光增强的程度与核酸的量成线性关系，据此实现了对核酸的定量测定。     

Bioanalytical applications of isothermal nucleic acid amplification techniques

The most popular in vitro nucleic acid amplification techniques like polymerase chain reaction (PCR) including real-time PCR are costly and require thermocycling, rendering them unsuitable for uses at point-of-care. Highly efficient in vitro nucleic acid amplification techniques using simple, portable and low-cost instruments are crucial in disease diagnosis, mutation detection and biodefense. Toward this goal, isothermal amplification techniques that represent a group of attractive in vitro nucleic acid amplification techniques for bioanalysis have been developed. Unlike PCR where polymerases are easily deactivated by thermally labile constituents in a sample, some of the isothermal nucleic acid amplification techniques, such as helicase-dependent amplification and nucleic acid sequence-based amplification, enable the detection of bioanalytes with much simplified protocols and with minimal sample preparations since the entire amplification processes are performed isothermally. This review focuses on the isothermal nucleic acid amplification techniques and their applications in bioanalytical chemistry. Starting off from their amplification mechanisms and significant properties, the adoption of isothermal amplification techniques in bioanalytical chemistry and their future perspectives are discussed. Representative examples illustrating the performance and advantages of each isothermal amplification technique are discussed along with some discussion on the advantages and disadvantages of each technique.     

Study on the interaction between nucleic acid and Eu3+-oxolinic acid and the determination of nucleic acid using the resonance light scattering technique

At pH 9.75, the resonance light scattering (RLS) intensity of OA–Eu³⁺ system is greatly enhanced by nucleic acid. Based on this phenomenon, a new quantitative method for nucleic acid in aqueous solution has been developed. Under the optimum condition, the enhanced RLS is proportional to the concentration of nucleic acid in the range of 1.0 × 10⁻⁹ to 1.0 × 10⁻⁶ g/ml for herring sperm DNA, 8.0 × 10⁻¹⁰ to 1.0 × 10⁻⁶ g/ml for calf thymus DNA and 1.0 × 10⁻⁹ to 1.0 × 10⁻⁶ g/ml for yeast RNA, and their detection limits are 0.020, 0.011 and 0.010 ng/ml, respectively. Synthetic samples and actual samples were satisfactorily determined. In addition, the interaction mechanism between nucleic acid and OA–Eu³⁺ is also investigated.     

Terbium fluorescence as a sensitive,inexpensive probe for UV-induced damage in nucleic acids

Much effort has been focused on developing methods for detecting damaged nucleic acids. However, almost all of the proposed methods consist of multi-step procedures, are limited, require expensive instruments, or suffer from a high level of interferences. In this paper, we present a novel simple, inexpensive, mix-and-read assay that is generally applicable to nucleic acid damage and uses the enhanced luminescence due to energy transfer from nucleic acids to terbium(III) (Tb³⁺). Single-stranded oligonucleotides greatly enhance the Tb³⁺ emission, but duplex DNA does not. With the use of a DNA hairpin probe complementary to the oligonucleotide of interest, the Tb³⁺/hairpin probe is applied to detect ultraviolet (UV)-induced DNA damage. The hairpin probe hybridizes only with the undamaged DNA. However, the damaged DNA remains single-stranded and enhances the intrinsic fluorescence of Tb³⁺, producing a detectable signal directly proportional to the amount of DNA damage. This allows the Tb³⁺/hairpin probe to be used for sensitive quantification of UV-induced DNA damage. The Tb³⁺/hairpin probe showed superior selectivity to DNA damage compared to conventional molecular beacons probes (MBs) and its sensitivity is more than 2.5 times higher than MBs with a limit of detection of 4.36 ± 1.2 nM. In addition, this probe is easier to synthesize and more than eight times cheaper than MBs, which makes its use recommended for high-throughput, quantitative analysis of DNA damage.     

Determination of nucleic acids based on the fluorescence quenching of Hoechst 33258 at pH 4.5

It was found the strong fluorescence emitted by the bis-benzimidazole derivative Hoechst 33258 at 490 nm could be efficiently quenched in pH 4.5 buffer when nucleic acids were added. Analysis of fluorescence intensity showed that the procedure was a static quenching dominated one, which was also demonstrated by the electron absorption spectra and lifetime of the excited state. The binding constant and numbers of binding sites were obtained from the Scatchard plot. The decreased fluorescence intensity was in proportion to the concentration of nucleic acids in the range 40-1800 ng ml⁻¹ for dsDNA and 26-1700 ng ml⁻¹ for ssDNA. The limits of detection were 12 and 8 ng ml⁻¹, respectively. The sensitivity of the method was about 3.4 times higher for dsDNA detection and 5.4 times higher for ssDNA detection compared with the widely used fluorescence enhancement method using the same dye. Application results to synthetic samples showed simplicity, rapidity and satisfactory reproducibility of the presented method. Measurement of real samples extracted from leaves of Crassula argentea and E. coli genome also gave satisfactory results, which were in good agreement with those obtained using spectrophotometric method.     

HPLC determination of carminic acid in foodstuffs and beverages using diode array and fluorescence detection

Summary Carmine extracted from cochineal insects is one of the most used natural colorings for beverages and other foods. Its active ingredient is carminic acid (7-β-D-glucopyranosyl-9,10-dihydro-3,5,6,8-tetrahydroxy-1-methyl-9,10-dioxo-2-anthracenecarboxylic acid). This work describes a rapid HPLC determination of carminic acid and compares diode array and fluorescence detections for quantification. Samples with higher protein levels, such as milk and yogurt, are first treated with 1 mL of 8 M NH₄OH (5 min), the pH is reduced to 2 with 6 M HCl before centrifugation, the supernatant is then filtered and injected into the chromatograph. Low protein samples are simply filtered before injection. The separations were performed with a LiChroCART RP18 column using a mixture of acetonitrile and formic acid as mobile phase. The optimized conditions permit baseline quantification of the carminic acid even in the presence of other coloring agents. The sampling and analytical procedures are considerably faster than those of the literature and present excellent recuperation, selectivity, accuracy and precision. The method was applied to analysis of several yogurts and beverages. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Utility of lab-on-a-chip technology for high-throughput nucleic acid and protein analysis

On-chip electrophoresis can provide size separations of nucleic acids and proteins similar to more traditional slab gel electrophoresis. Lab-on-a-chip (LoaC) systems utilize on-chip electrophoresis in conjunction with sizing calibration, sensitive detection schemes, and sophisticated data analysis to achieve rapid analysis times (<120 s). This work describes the utility of LoaC systems to enable and augment systems biology investigations. RNA quality, as assessed by an RNA integrity number score, is compared to existing quality control (QC) measurements. High-throughput DNA analysis of multiplex PCR samples is used to stratify gene sets for disease discovery. Finally, the applicability of a high-throughput LoaC system for assessing protein purification is demonstrated. The improvements in workflow processes, speed of analysis, data accuracy and reproducibility, and automated data analysis are illustrated.     

Polypyrrole-polyvinyl sulphonate film based disposable nucleic acid biosensor

Double stranded calf thymus deoxyribonucleic acid entrapped polypyrrole-polyvinyl sulphonate (dsCT-DNA-PPy-PVS) films fabricated onto indium-tin-oxide (ITO) coated glass plates have been used to detect organophosphates such as chlorpyrifos and malathion. These disposable dsCT-DNA-PPy-PVS/ITO bioelectrodes have been characterized using cyclic voltammetry, Fourier-transform-infra-red (FTIR) spectroscopy and atomic force microscopy (AFM), respectively. These biosensing electrodes have a response time of 30 s, are stable for about 5 months when stored in desiccated conditions at 25 °C and can be used to amperometrically detect chlorpyrifos (0.0016-0.025 ppm) and malathion (0.17-5.0), respectively. The additive effect of these pesticides on the amperometric response of the disposable dsCT-DNA-PPy-PVS/ITO bioelectrodes has also been investigated.     

First improvements in the detection and quantification of label-free nucleic acids by laser-induced breakdown spectroscopy: Application to the deoxyribonucleic acid micro-array technology

The accurate quantification of nucleic acids is essential in many fields of modern biology and industry, and in some cases requires the use of fluorescence labeling. Yet, in addition to standardization problems and quantification reproducibility, labeling can modify the physicochemical properties of molecules or affect their stability. To address these limitations, we have developed a novel method to detect and quantify label-free nucleic acids. This method is based on stoichiometric proportioning of phosphorus in the nucleic acid skeleton, using laser-induced breakdown spectroscopy, and a specific statistical analysis, which indicates the error probability for each measurement. The results obtained appear to be quantitative, with a limit of detection of 10⁵ nucleotides/µm² (i.e. 2 × 10¹³ phosphorus atoms/cm²). Initial micro-array analysis has given very encouraging results, which point to new ways of quantifying hybridized nucleic acids. This is essential when comparing molecules of different sequences, which is presently very difficult with fluorescence labeling.     

Adsorption of nucleic acid bases on clays: an investigation using Langmuir and Freundlich isotherms and FT-IR spectroscopy

In the present paper, the adsorption of nucleic acid bases (A, adenine; C, cytosine; U, uracil; and T, thymine) on clays (bentonite, kaolinite, and montmorillonite) was studied at different pH (2.00 and 7.20). It should be pointed out there is no reported study of adsorption of nucleic acid bases on clays using seawater (with the major elements), and a wide range of pH. The main finding of this study was that the ratio of A and T adsorbed on clays ranged from 4.68 to 25.1, much higher than the ratio of their occurrence in organisms ranging from 0.95 to 1.05. The weaker adsorption of U and T on clays raises the question of the possibility of a genetic code based on purines only. The FT-IR spectra at pH 2.00 showed that the interaction of A, C, T, and U with the clays occurs through positively charged, protonated groups. Correspondence: Dr. Dimas A. M. Zaia, Departamento de Química-CCE, Universidade Estadual de Londrina, 86051-990 Londrina-PR, Brazil.